Osteoporosis is a clinical condition of decreased bone mass secondary to aging, menopause, metabolic dysfunction, and decreased mechanical loading. Elucidating factors that maintain and build bone mass is essential for development of novel treatments for these devastating conditions of bone loss. One promising anabolic pathway to further explore is canonical Wnt signaling. Canonical Wnt signaling potently induces the differentiation of progenitor cells to become bone-forming osteoblasts. The sponsor's laboratory recently demonstrated that the Wnt co-activator R-spondin 2 (RSPO2) can also promote osteoblast differentiation in vitro. R-spondins were recently established to signal through the transmembrane proteins Leucine-rich repeat- containing G-protein coupled Receptors 4, 5, 6 (LGR4-6); however, current research is unclear on whether this is the predominant signaling mechanism for RSPO2, particularly in osteoblasts. This proposal will determine whether RSPO2 operates through the Wnt signaling pathway and LGR receptors. As well, the translational question of whether increasing Rspo2 in osteoblasts in vivo is sufficient to increase bone mass will be addressed. First, changes in Wnt signaling pathway activation relative to RSPO2 levels during osteoblast differentiation will be defined. The necessity of canonical Wnt signaling activit for RSPO2 action in osteoblasts will be determined through inhibition of canonical Wnt signaling. Next, the requirement of LGRs for RSPO2 action in progenitor cells will be investigated by knockdown of the receptors. The expression pattern of LGRs in bone as well as changes in LGR expression during osteoblastogenesis will also be examined. Finally, Rspo2 will be conditionally overexpressed in osteoblasts in mice, and long bones will be evaluated for changes in bone mass, bone formation rate, and osteoblast differentiation capabilities of progenitor cells. Additionally, this mouse line will be crossed with a TOPGAL mouse line to assess changes in Wnt signaling activation in vivo due to Rspo2 overexpression. These experiments are critical steps in advancing the understanding of osteoblastogenesis and in harnessing the Wnt signaling pathway to design anabolic therapeutics for osteoporosis treatment and prevention.